Current Pharmaceutical Analysis, 2007, 3, 117-128 117 1573-4129/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. Hyaluronan Determination: Biological Significance & Analytical Tools Christina J. Malavaki 1 , Ioannis Kanakis 1 , Achilleas D. Theocharis 1 , Fotini N. Lamari 2 and Nikos K. Karamanos 1, * 1 Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece; 2 Department of Pharmacy, University of Patras, Patras, Greece Abstract: Hyaluronic acid is a glycosaminoglycan, which is one of the main components of the extracellular matrix, contributes signifi- cantly to cell proliferation and migration, and is involved in many physiological and pathological biologic processes, such as the progres- sion of some malignant tumors. Therefore, the determination of its amounts may be of use for monitoring the progress of some diseases and/or as prognostic/diagnostic marker. Hyaluronan has been used for the therapy of osteoarthritis, for ophthalmic and cosmetic surger- ies, and is under investigation for numerous other diseases. In this review, after a short introduction to hyaluronan structure and biologic roles, the electrophoretic, chromatographic and solid-phase assays used for determination of its concentration in biologic samples and in drug formulations are presented. Keywords: Hyaluronic acid, Biological roles, Analysis, Electrophoresis, Chromatography, Solid-phase assay. STRUCTURE AND BIOLOGICAL ROLES OF HA Hyaluronic acid (HA), commonly referred to as hyaluronan, is a negatively charged, non-sulfated linear glycosaminoglycan (GAG), consisting of repeating units of ( 1 4)-D-glucuronic acid-( 1 3)- N-acetyl-D-glucosamine (Fig. 1). HA occurs typically as a polymer of high relative molecular mass (Mr) (up to 2x10 4 kDa) in the ex- tracellular matrix of almost all animal tissues existing in significant quantities in the skin, synovial fluid, brain and central nervous sys- tem, and the vitreous body of the eye [1,2]. In contrast with all other GAGs, HA is not linked to a core protein and it is not synthe- sized in the Golgi apparatus, but at the cytoplasmic surface of the plasma membrane [3]. Multi-drug resistance transporter system is possibly involved in the transportation of HA out of the cell as it is being synthesized [4]. There are four HA synthase (HAS) genes in most vertebrate ge- nomes with only three in mammals. HAS are glycosyl transferases which differ in catalytic rates and synthesize HA of different mo- lecular masses. HAS-1 is the least active and produces HA of high Mr (2x10 2 - 2x10 4 kDa), HAS-2 is catalytically more active and synthesizes HA of similar size and might be the main enzyme, which produces HA in stress-induced conditions and wound heal- ing. HAS-3 is the most active enzyme, which produces HA of lower Mr. Expression of the HAS genes seems to be tissue specific and their expression is controlled in part by various growth factors and cytokines [5]. * Address correspondence to this author at the Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26 110 Patras, Greece; Tel: +30-2610-997915; Fax: +30-2610-997153; E-mail: N.K.Karamanos@upatras.gr The degradation of HA is mediated through binding of HA to its cell surface receptors CD44 and RHAMM, internalization and degradation within cells [6, 7]. Two hyaluronidases (Hyals) are involved in the catabolism. HA polymers are bound to the cell membrane through the combined action of CD44 and Hyal2 [8]. Hyal2 is glycosylphosphatidylinositol (GPI)-anchored at the exter- nal surface of the plasma membrane, and initiates cleavage of high Mr HA [9]. Fragments of 20 kDa are then delivered to the early endosomes and to lysosomes where degradation continues through the action of acid-active Hyal1 [10]. HA may be released from tissue matrices into the vasculature and lymphatic system with final location the liver, kidney and pos- sibly spleen. These pathways involve unique receptors, such as HA receptor for endocytosis (HARE) [11], lymphatic vessel endothelial HA receptor (LYVE)-1 [12] and layilin [13]. HA may also be fragmented by free radicals under oxidative conditions. Free radi- cals and Hyals are coordinated under certain pathologic conditions [8]. HA of high Mr is highly hygroscopic occupying a large volume in solution and acts as a space-filling molecule hydrating tissue and regulating osmotic balance. HA is able to interact with various ma- trix and cell surface molecules, such as CD44 and hyaladherins, participating in tissue organization and integrity [14]. HA directly regulates viscoelasticity, which imparts weight-bearing capacity to tissues, such as cartilaginous joints, as well as lubrication during their movement [2]. High Mr HA is also anti-inflammatory and immunosuppresive [15]. The latter is partly attributed to the ability of HA to coat cell surfaces and to prevent ligand access to surface receptors. High Mr HA is involved in the entire process of ovulation, fertilization and embryogenesis [8]. It also inhibits phagocytosis by monocytes, macrophages and peripheral mononuclear cells [16]. HA of high Mr promotes tissue integrity and quiescence, while fragments of HA derived usually through degradation process signal that injury has occurred. HA of low Mr is elevated at sites of injury and is medi- ated through mechanisms involving activation of Hyals, chondroit- inases and hexosaminidases and free radicals. The low Mr HA stimulates the expression of inflammatory genes including TNF-a, IGF-I, interleukin-1beta and interleukin-8 [17]. When injury occurs, HA of high Mr accumulates that is anti- angiogenic and immunosuppressive, making a loose tissue, which facilitates peripheral mononuclear cell access to the wound site for removal of dead tissue, debris and bacteria [8]. The next phase of wound healing is characterized by increase of low Mr HA, which triggers a cascade of biological process necessary for tissue repair. Fig. (1). Structure of hyaluronan repeating disaccharide unit. O O O HO ABC/AC CH 2 OH NH C O CH 3 O COOH O HO OH n